Journal of Biomechanical Science and Engineering
Online ISSN : 1880-9863
ISSN-L : 1880-9863
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Analysis of membrane structure of the inner ear motor protein prestin by force spectroscopy
Michio MURAKOSHIHiroshi WADA
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JOURNAL OPEN ACCESS

2021 Volume 16 Issue 3 Pages 21-00125

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Abstract

The high sensitivity of mammalian hearing is achieved by cochlear amplification. The basis of this amplification is the motility of outer hair cells (OHCs), which are sensory cells in the inner ear. This motility may be due to voltage-dependent conformational changes of the motor protein prestin, which is densely embedded in the lateral membrane of OHCs. However, the membrane structure of prestin has not yet been elucidated. Therefore, the membrane structure of prestin was herein investigated by force spectroscopy using an atomic force microscope (AFM). The gene of prestin fused with an Avi-tag at its C terminus was transfected into Chinese hamster ovary (CHO) cells and the inside-out plasma membrane was isolated. The Avi-tag was enzymatically biotinylated and attached to a streptavidin-coated AFM cantilever via biotin-streptavidin binding. Prestin was then pulled out from the plasma membrane and the relationship between the force applied to the protein and the extension distance, i.e., the force-extension (FE) curve, was assessed. The curves obtained showed saw-toothed patterns. An attempt was then made to analyze these curves using the worm-like chain model. The force caused by stretching of the intracellular C terminus and that due to the extraction of one or several transmembrane domains were identified. The present results imply that the C terminus and the subsequent transmembrane domains of prestin correspond to those of the previously reported model with 12 transmembrane domains.

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© 2021 by The Japan Society of Mechanical Engineers

This article is licensed under a Creative Commons [Attribution 4.0 International] license.
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